Team:Slovenia/METHODS and PARTS/protocols/bm

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Cloning & Proteins Binding Assays Biosynthesis Methodology Split/FRET Methodology protocols - Biosynthesis Methodology

SYNTHESIS OF VIOLACEIN

 

Overnight cultures of E. coli containing plasmids encoding zinc finger fusion proteins for violacein synthesis with or without plasmid encoding DNA program were diluted 1:1000 in fresh Luria Bertani broth and grown at 30°C in the presence of appropriate antibiotics. At various time points samples were taken. Bacteria were lysed by addition of equal volume of 10% SDS, and violacein extracted with 1:1 (v/v) ethylacetate. After brief vortexing, the organic phase was collected and absorbance spectrum (575 nm) was measured. To better separate and quantify the amount of violacein and deoxyviolacein, samples were further analyzed by HPTLC and mass spectroscopy. The quantity of violacein or deoxyviolacein production with or without DNA program or in the presence of scrambled DNA program was compared.

 

 

TLC

 

Thin layer chromatography (TLC) is a chromatography technique used to separate mixtures. Thin layer chromatography is performed on a sheet of glass, plastic, or aluminum foil, which is coated with a thin layer of adsorbent material. This layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved. Violacein standard (0.01 mg/ml) in methanol was applied with samples on HPTLC silica gel plates (20 x 10 cm) using Automatic TLC Sampler 4 (Camag, Muttenz, Switzerland). Plates were developed in 4 min simultaneously from both sides up to 4.5 cm from the bottom of the plate in dichloromethane:methanol (6:1, v/v) using a horizontal developing chamber (sandwich configuration). The chromatograms were evaluated quantitatively at 570 nm in absorption/reflectance mode by using TLC Scanner 3 (Camag).

 

 

MASS SPECTROMETRY

 

Violacein was also identified by LCQ mass spectrometer (Thermo Finnigan, San Jose, CA, USA) coupled to TLC-MS interface (Camag) from the plate using electrospray ionization in a positive mode. The quantity of violacein production with or without nucleic acid program or in the presence of scrambled nucleic acid program was compared.

 

 

SYNTHESIS OF CAROTENOIDS

 

Overnight cultures of E. coli containing plasmids encoding zinc finger fusion protein for astaxantin synthesis  with or without plasmid encoding DNA program were diluted 1:1000 in fresh Luria Bertani broth and grown at 30^oC in the presence of appropriate antibiotics. At various time points samples were taken and bacteria were removed by sedimentation (5 min at 13.000 rpm). To facilitate bacterial lysis, bacterial pellets were a freeze-thawed at -80^oC room temperature, respectively. Carotenoids were extracted with mixture of organic solvents.

 

 

HPLC

 

High-performance liquid chromatography (or high-pressure liquid chromatography, HPLC) is a chomatographic technique that can separate a mixture of compounds and is used to identify, quantify and purify the individual components of the mixture. HPLC typically utilizes different types of stationary phase (hydrophobic) saturated carbon chains, a pump that moves the mobile phase(s) and analyte through the column, and a detector that provides a characteristic retention time for the analyte. Samples of carotenoids were then analyzed by HPLC-UV system consisted of LC pump P2000 (Thermo, Finnigan, San Jose, CA, USA), autosampler Spectrasystem TPS (AS 100) with fixed 20 µl loop and UV detector (Thermo, Finnigan). Separation was performed on a stainless-steel column Prontosil C30, (250 x 4.6 mm I.D.) with Phenomenex HPLC guard cartridge C18 as a precolumn. Acetone or acetone:water (9:1, v/v) were used as a mobile phase for beta-carotene or lycopene and zeaxanthin or canthaxanthin, respectively. All compounds were detected at 450 nm. Flow rate was 1 ml/min for all compounds while run time was 15 min for beta-carotene or lycopene and 25 min for zeaxanthin or canthaxanthin.